The T Box Mechanism and Anticodon Stem-Loops: Molecular and Structural Studies of Glycyl-tRNA Anticodon Stem-Loops and Their Binding to the T Box Specifier Domain.

Author

Chang, Andrew

Date

2013-11-22

Advisor

Nikonowicz, Edward P.

Degree

Doctor of Philosophy

Abstract

T box mechanism is a riboswitch commonly used by Gram-positive bacteria to regulate expression of amino-acid related genes such as aminoacyl-tRNA synthetases (aaRS). The T box riboswitch regulates the gene by the mechanism of transcription attenuation. The 5’-UTR of the mRNA forms mutually exclusive anti-terminator or terminator structures depending on whether the tRNA bound is uncharged or charged. This study focuses on the interactions that occur between T box specifier domain (SD) and tRNA anticodon stem-loop (ASL). This intermolecular interaction contributes to the specificity of the T box riboswitch. In bacteria, glycyl-tRNA molecules with anticodon sequences GCC and UCC exhibit multiple extratranslational functions, including transcriptional regulation and cell wall biosynthesis. In this study, the high-resolution structures of three glycyl-tRNA anticodon arms with anticodon sequences GCC and UCC have been determined. Two of the tRNA molecules are proteinogenic and one is non-proteinogenic and it participates in cell wall biosynthesis. The structures of the three tRNAGly anticodon arms exhibit small differences between one another and there is no evidence that they form the canonical U-turn motif. The Specifier domain of the T box riboswitch contains the Specifier sequence that is complementary to the tRNA anticodon and is flanked by a highly conserved purine nucleotide that could result in a fourth base pair involving the invariant U33 of tRNA. We show that the interaction between the T box Specifier domain and tRNA consists of three Watson–Crick base pairs and that U33 confers stability to the complex through intramolecular hydrogen bonding. The NMR data also suggests the ASL may change its structure to form a U-turn when in complex with the T box Specifier domain. Other T box domains also have specific tertiary structure. The NMR data in this study supports the fact that the T box apical loop interacts with the AG loop as seen in the crystal structure. The NMR data suggests that the U70 of the apical loop forms a reversed Hoogsteen A-U base pair with A73 and a U70G mutation has detrimental effects on the structures and interactions between the two loops.